Automated quantification of DNA strand breaks in intact cells

ABSTRACT

The present invention relates to an automated method of quantifying DNA strand breaks in intact cells whereby 
     (a) cells are lysed in order to release DNA, 
     (b) the lysate is subjected to an alkaline denaturation process, 
     (c) neutralization occurs, 
     (d) a fluorescent dye is added, and 
     (e) fluorescence is read off a fluorometer, 
     and the method is characterized in that all pipetting stages are carried out in automated fashion by means of a pipetting device in a lightproof housing. The invention further relates to a device to carry out said method, comprising a lightproof housing, a temperature controlled base plate, a system of pipetting nozzles and a fluorometer to measure the fluorescent intensity of DNA.

This application claims priority to the German Patent Application 1 9643 721.0, filed on Oct. 23, 1996.

I. FIELD OF THE INVENTION

The present invention relates to an automated method of quantifying DNAstrand breaks in intact cells and a device to carry out the method.

II. BACKGROUND OF THE INVENTION

The quantitative detection of DNA strand breaks is of great significancefor many fields of biomedical research and diagnosis already today. Themechanisms of DNA-damage and DNA repair and their disturbances can beinvestigated via the detection of DNA strand breaks. Furthermore, it isthereby possible to carry out toxicological screening of substances suchas chemicals, natural substances or pharmaceutical preparations.Moreover, various cell and tissue samples from patients can be examinedfor DNA damage and DNA repair capacity, respectively, by quantifying DNAstrand breaks. This is of interest for what is called monitoring theeffectiveness of radiation therapy and cytostatic chemotherapy,respectively, in the malignant cells to be killed before and during thetreatment. It is also possible to evaluate the side-effect risk ofradiation therapy and chemotherapy, respectively, before the therapy isstarted by examining normal (nonmalignant) cells as to DNA strandbreaks. The examination of DNA strand breaks is also significant withinthe scope of preventive medicine for the screening for individualshaving a high degree of DNA damage and low DNA repair capacity,respectively. A greater cancer risk has to be assumed for these persons,so that an especially close-knit preventative program is indicated forthem. In this connection, what is called biomonitoring must also beconsidered in industrial medicine. Here, standardized DNA damagetreatments would each be made with the proband's cell material to betested.

So far, there have been some methods of examining DNA strand breaks,which are, however, very time-consuming and labor-intensive, since theymust mostly be carried out manually. Another drawback of these methodsis the care which must urgently be taken in the various steps so as notto reach false-positive or false-negative results. The “alkalineelution” has to be mentioned as a known method of examining DNA strandbreaks. Here, a controlled, alkaline denaturation of the DNA is inducedin a cell lysate. The strand break frequency is measured by determiningthe elution rate of the single-stranded DNA fragments through a suitablepolycarbonate filter. Another known method is what is called the “cometassay”. This is an in situ assay which can be carried out in variousways. In this case, cells must be embedded in an agarose gel. Followinglysis “in situ” an electric field is applied, which results in a more orless marked migration of chromatin out of the nucleus. Themicroscopically readable migration path is considered a standard thatapplies to the number of DNA strand breaks. In addition, the“Fluorescence-detected Alkaline DNA Unwinding” (Birnboim, H. C. andJevcak, J. J., 1981, Cancer Res. 41:1889-1892, abbreviated as“FADU-Assay”, is also known for measuring DNA strand breaks. Here, testcells are lysed and the cellular DNA exposed in this way, whichdepending on the pretreatment of the cells has a more or less largenumber of single-strand or double-strand breaks is then subjected todenaturation under accurately controlled conditions, whereby the DNAdouble strand is converted into single strands. In practice, this iseffected by an extremely careful piling-up of an alkaline solution, eachmixture with the lower phase (the lysate) having to be avoided by allmeans. Within some minutes, part of the alkaline solution diffuses intothe lysate. The alkaline denaturation then starts in each case from theDNA break ends and chromosome ends, respectively, and proceeds linearly,namely in both orientations in the case of a single-strand break. Afterstopping this alkaline DNA-unwinding by neutralization, the rest of thenon-denatured DNA which remains in the sample is measured withnon-denatured DNA via the intensity of the ethidium bromidefluorescence. The amount of denatured DNA calculated therefrom is adirect standard that is applied to the number of DNA strand breakspresent at the time of cell lysis. The fluorescence measurement isstandardized so that it applies (i) to those cell lysates (referred toas “T samples”) in which no denatured pH was reached because theneutralization buffer had been added prior to the addition of thealkaline solution and (ii) to those lysates (referred to as “B samples”)which had been provided with a very large number of DNA breaks by DNAshearing (e.g., by means of ultrasound treatment) prior to the alkalinedenaturation. In the case of the T sample the content of denatured DNAis set to 0%, whereas the content of denatured DNA in the B sample isset to 100%. However, the FADU assay is very labor-intensive andsusceptible to failure because several high-precision pipetting stepsand the accurate observance of time and temperature conditions arerequired for each individual sample. In some steps attention has to bepaid to the fact that the contents of the sample tubes comprisingseveral piled-up liquid phases are not mixed. With regard to therequired quadruple parallel determinations, the T and B samples whichalways have to be carried along and the large number of samples to bedetermined this adds up to an immense pipetting work, each pipettingstep having to be carried out with the utmost constant care. In thisconnection, it is aggravating that the FADU assay must be carried outwhile the laboratory is darkened, since the cell lysates are verysensitive to light.

As explained already, the drawback of all of these methods is that theyare very labor-intensive and the sample throughput per time unit is notvery high. Thus, about 3 manhours are required to manually carry out theFADU assay with 30 samples. As regards the alkaline elution 16 hours 2of them manhours) have to be estimated for about 36 samples.

In the case of the comet assay even 8 manhours are required to processonly 20 samples.

Therefore, it is the object of the present invention to provide a methodof quantifying DNA strand breaks, which can be carried out easily, has agood sample throughput per time unit and supplies reliable results. Theobject of the present invention also consists in providing a device bymeans of which the method can be carried out.

III. SUMMARY OF THE INVENTION

The present invention relates to an automated method of quantifying DNAstrand breaks in intact cells and a device to carry out the method.

IV. DETAILED DESCRIPTION OF THE INVENTION

It is the object of the present invention to provide a method ofquantifying DNA strand breaks, which can be carried out easily, has agood sample throughput per time unit and supplies reliable results. Theobject of the present invention also consists in providing a device bymeans of which the method can be carried out.

This object is achieved by a method according to claim 1. Furthermore,this object is achieved by a device according to claim 5. Advantageousembodiments follow from the dependent claims.

The inventor recognized that there was a demand for standardizing theexamination of DNA strand breaks. This is achieved by the highestpossible automation of the method.

The method according to the invention distinguishes itself in that

(a) the cells are lysed in order to release DNA,

(b) the lysate is subjected to an alkaline denaturation process,

(c) neutralization occurs,

(d) a fluorescent dye is added, and

(e) the fluorescence is read off a fluorometer,

all of the pipetting stages being carried out in automated fashion bymeans of a pipetting device in a lightproof housing and preferably thefluorescence measurements are also carried out in automated fashion.

The method according to the invention regarding what is called the “P”samples comprises preferably the following individual steps, thepipetting stages being carried out by means of an automatic pipettingdevice (“pipetting robot”):

1) placing a reaction vessel, e.g., a microtiter plate, containingadherent cells in cell culture medium or suspension cells in medium,preferably in solution B, in a darkened pipetting station

2) in the case of adherent cells: drawing off the medium; incubationtemperature 0° C.; then immediately

3) in the case of adherent cells: adding solution B (0° C.); incubationtemperature 0° C., then immediately

4) adding solution C (room temperature); incubation temperature 0° C.;5-20 minutes (preferably 10 minutes)

5) adding solution D (0° C.) (must be piled up without being vortexed);incubation temperature 0° C.; then immediately

6) adding solution E (0° C.) (must be piled up without being vortexed);incubation temperature 0° C.; 15-45 minutes (preferably 30 minutes)

7) adjusting the incubation temperature to about 15° C.; 30-90 minutes(preferably 60 minutes)

8) adding solution F (0° C.); mixing; incubation temperature 0° C.; 5-15minutes (preferably 10 minutes)

9) shearing the sample, e.g., by rapid pipetting up and down orultrasonic treatment,

10) adding solution G (room temperature); incubation temperature about20° C.; 5-20 minutes (preferably 10 minutes)

11) reading the fluorescence intensities

12) data evaluation.

The solutions employed are, e.g., those mentioned in Bimboim et al.,1981, Cancer Res. 41:1889-1892. In detail these are:

Solution B: 0.25 M meso—inositol—10 mM sodium phosphate—1 mM MgCl₂ (pH7.2)

Solution C: 9 M urea—10 mM NaOH—2.5 mM cyclohexane diaminetetraacetate—0.1% sodium dodecylsulfate (=lysis buffer; storage at roomtemperature)

Solution D: 0.45 volume of solution C in 0.20 N NaOH (=alkaline solutionI)

Solution E: 0.40 volume of solution C in 0.20 N NaOH (=alkaline solutionII)

Solution F: 1 M glucose—14 mM 2-mercaptoethanol (=neutralizationsolution)

Solution G: ethidium bromide 6.7 μg/ml—13.3 mm NaOH (storage at roomtemperature)

The above-mentioned times, temperatures and solutions can, of course,vary within certain limits. These variations are within the skill of aperson skilled in the art and can be determined by means of routineexperiments.

In a preferred embodiment, solutions D and E are applied combined in onestep instead of two steps (steps 5) and 6)).

In the operating cycle for samples in which the content of denatured DNAcan be set to 0% (referred to as “T samples” above), step 8) directlyprecedes step 5) already. In the operating cycle for sample in which thecontent of denatured DNA can be set to 100% (referred to as “B samples”above), step 9) directly precedes step 7) already.

The percentage D of double-stranded DNA which is still found in thesample following the partial alkaline treatment is a standard that isapplied to the number of strand breaks at the time of lysis and can becalculated as follows:

D(%)=(P−B)./.(T−B)×100

In place of ethidium bromide which is known to be mutagenic, it is alsoin principle possible to use another non-toxic fluorescent dye which isintercalated in double-stranded DNA (e.g., Sybr-Green™) in solution G.

The expression “adherent cells” refers to those cells which can becultured in a reaction vessel, e.g., on a microtiter plate, as amonolayer in cell culture medium. Suitable cell culture mediums are thepurchasable media with which the person skilled in the art is familiarand which are selected depending on the cell type to be grown. Somehours to about one day later the cells grown on the reaction vessel canbe placed directly in the device. Examples of adherent cells arefibroblasts, HeLa cells and most tumor cells.

However, it is also possible to use “suspension cells”. Suspension cellsare cells which cannot be cultured as monolayer on cell culture plates.The suspension cells must first be centrifuged off the correspondingmedium and then be resuspended in a buffer, e.g., in solution B, beforethey can be filled in a reaction vessel, e.g., a microtiter plate, andbe examined according to the above scheme. Examples of suspension cellsare blood cells, such as leukocytes, or lymphoid cell lines.

In a preferred embodiment, the process according to the invention canalso be extended to the quantification of certain damage of DNA bases.To this end, the cell lysate which was treated with a lysis buffercontaining less urea than solution C is subjected to an incubation usinga purified DNA repair endonuclease prior to the alkaline denaturatin,which recognizes DNA damage determined in a highly specific way (e.g.,8-oxaguanine and pyrimidine dimers, respectively) and cuts locally theaffected DNA strands, so that now one DNA strand break forms for eachdamaged base. These additional breaks generated in vitro can then bedetermined immediately afterwards by the measuring method according tothe invention. An example of such an endonuclease is what is called the“FAPY enzyme”. By this, the field of application of the method accordingto the invention can be extended considerably, namely to the highlyspecific quantification of unrepaired primary damage in DNA bases.

The device according to the invention is in principle a pipetting robotwhich comprises a lightproof housing. The reaction vessel, preferablythe microtiter plate, is placed therein on a temperature controlled baseplate which can raise the temperature of the samples from 0° C. to 30°C. The device comprises a system of pipetting nozzles for withdrawingliquids from, and adding them to, respectively, the reaction vessel,preferably the microtiter plate. Another component of the device is afluorometer having an excitation wavelength of 490-550 nm, preferably520 nm, and an emission wavelength of 570-610 nm, preferably 590 nm. Thefluorometer is equipped such that the fluorescence intensity in eachwell of the preferred microtiter plate can be read in parallel orsequentially “in situ”. The expression “fluorometer” also comprises asimple device permitting the observation of the fluorescence phenomenonby means of a light source and two different filters. The deviceaccording to the invention also comprises preferably a microprocessor aswell as a printer, and plotter, respectively.

Advantages of the method according to the invention are represented bythe fact that by using the device according to the invention amicrotiter plate having 96 measuring points can be treated in less than2 device hours. In contrast thereto, the working time for the staff isonly several minutes as compared to the above-mentioned periods requiredfor the manual examination of DNA strand breaks. Moreover, the celllysis can be made directly with adherent single-layer cell cultures bythe method according to the invention. The latter is of special interestbecause by this the preceding removal of cells from the substrate (e.g.,by trypsin) is not necessary. Thus, corresponding centrifugation stepscan be omitted, which are (i) time-consuming and costly and (ii) canartificially already result in the induction of DNA breaks andunfavorably affect the DNA repair behavior of the cell, respectively.Thus, the method according to the invention offers the additionaladvantage of measuring the DNA damage and repair in an unadulteratedform.

The below examples explain the invention in more detail. The followingpreparations and examples are given to enable those skilled in the artto more clearly understand and to practice the present invention. Thepresent invention, however, is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only, and methods which are functionally equivalent arewithin the scope of the invention. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

V. EXAMPLE

HeLa cells are placed with DMEM medium (Dulbelcco's modified essentialmedium) plus 10% fetal calf serum in the wells of a microtiter platesuitable for cell culture and incubated for 12 hours, the cells growingcompletely. Thereafter, the intact cells are exposed to radioactiveradiation (⁶⁰Co γ radiation) at 0° C. (on ice). Then, the methodaccording to the invention is carried out as follows:

1) placing the microtiter plate in the darkened pipetting station,

2) drawing off the medium; temperature of the base plate 0° C.; thenimmediately

3) adding 40 μl of solution B (0° C.); temperature of the base plate 0°C., then immediately

4) adding 40 μl of solution C (room temperature); temperature of thebase plate 0° C.; incubation for 10 minutes

5) adding 20 μl of solution D (0° C.) (must be piled up without beingvortexed); temperature of the base plate 0° C.; then immediately

6) adding 20 μl of solution E (0° C.) (must be piled up without beingvortexed); temperature of the base plate 0° C.; 15-45 minutes(preferably 30 minutes)

7) heating the base plate to about 15° C.; 60 minute of incubation

8) adding 80 μl of solution F (0° C.); mixing; temperature of the baseplate 0° C.; incubation for 10 minutes

9) shearing the sample, e.g., by rapid pipetting up and down

10) withdrawing 100 μl and transferring it to a new microtiter plate

11) adding 150 μl of solution G (room temperature); temperature of thebase plate about 20° C.; incubation for 10 minutes

12) reading the fluorescence intensities by means of the installedfluorometer (excitation wavelength 520 nm; emission wavelength 590 nm)

13) data evaluation.

The compositions of solutions B to G are indicated as outlined above.

In the operating cycle for samples whose content of denatured DNA shallbe set to 0% (referred to as “T samples” above) step 8) directlyprecedes step 5). In the operating cycle for samples whose content ofdenatured DNA can be set to 100% (referred to as “B samples” above) step9) directly precedes step 7.

In order to check the reliability of the method, all values weredetermined four times and the difference from one another wascalculated. This data evaluation resulted in the fact that thedeterminations yielded reliable values, the differences of the fourdeterminations being around 2%.

All references cited within the body of the instant specification arehereby incorporated by reference in their entirety.

What is claimed:
 1. A method of quantifying strand breaks in the DNA ofcells, comprising: (a) lysing cells in order to release DNA, whereby acell lysate is generated; (b) exposing said cell lysate to an alkalinedenaturation process, whereby said cell lysate is neutralized; (c)contacting said cell lysate with a fluorescent dye; and (d) measuringthe fluorescence from said fluorescent dye in contact with said celllysate, using a fluorometer; wherein steps (a) to (d) are carried out inautomated fashion utilizing a pipetting device in a lightproof housing.2. The method of claim 1, wherein said method takes place in microtiterplates.
 3. The method of claim 1, wherein said fluorescent dyeintercalates in double-stranded DNA.
 4. The method of claim 1, whereinsaid fluorescence is measured at an excitation wavelength of 520 nm andan emission wavelength of 590 nm.
 5. A device to carry out the method ofclaim 1, wherein said device comprises a lightproof housing, atemperature controlled base plate, a system of pipetting nozzles and afluorometer to measure the fluorescence intensity of DNA.
 6. The deviceof claim 5, wherein said device further comprises a microprocessor. 7.The device of claim 5, wherein the temperature controlled base plate isadjustable to temperatures from 0 to 30° C.
 8. The device of claim 5,wherein the fluorometer measures at an excitation wavelength of 520 nmand an emission wavelength of 590 nm.
 9. The method of claim 3, whereinsaid fluorescent dye is ethidium bromide.
 10. The device of claim 6,wherein said device further comprises a printer.
 11. The device of claim6, wherein said device further comprises a plotter.
 12. The method ofclaim 1, wherein said fluorescence is measured at an excitationwavelength in the range 490-550 nm and an emission wavelength in therange 570-610 nm.
 13. The device of claim 5, wherein the fluorometermeasures at an excitation wavelength in the range 490-550 nm and anemission wavelength in the range 570-610 nm.
 14. The method of claim 2wherein said microtiter plates have 96 measuring points.
 15. The methodof claim 1 wherein the cells are adherent.